Abstract
Ammonium can be transported into the cell by ion pumps in the cytoplasmic membrane. Ammonia then diffuse out through the cell membrane. A futile cycle is created that results in cytoplasmic acidification and extracellular alkalinisation. Ammonium transport can be quantified by measuring the extracellular pH changes occurring in a cell suspension (in PBS) after addition of ammonium. By using this technique, in combination with specific inhibitors of various ion pumps, it was shown that ammonium ions are transported across the cytoplasmic membrane by the Na+K+2Cl--cotransporter in both hybridoma and myeloma cells. Further, the Na+/H+ exchanger, which regulates intracellular pH by pumping out protons, was shown to be active during ammonium exposure. The viability of hybridoma cells suspended in PBS and exposed to NH sup+inf4 for only 90 min, was reduced by 11% (50% necrosis and 50% apoptosis). A control cell suspension did not loose viability during this time. Turning off the activity of the Na+/H+ exchanger (by amiloride) during ammonium exposure decreased viability further, while inhibiting transport itself (by bumetanide) restored viability to the same level as for the control experiment with bumetanide alone. These results show that one effect of ammonia/ammonium on cell physiology is specifically related to the inward transport of ammonium ions by membrane bound ion pumps.
Similar content being viewed by others
Abbreviations
- q pH :
-
specific rate of pH increase (pH units per min and 106 cells per ml)
References
Aronson PS, Suhm MA and Nee J (1983) Interaction of external H+ with the Na+-H+ exchanger in renal microvillus vesicles. J. Biol. Chem. 258: 6767–6771.
Borys MC, Linzer DIH and Papoutsakis ET (1994) Ammonia effects the glycosylation patterns of recombinant mouse placental lactogen-1 by chinese hamster ovary cells in a pH-dependent manner. Biotechnol. Bioeng. 43: 505–514.
Docherty PA and Snider MD (1991). Effects of hypertonic and sodium-free medium on transport of a membrane glycoprotein along the secretory pathway in culture mammalian cells. J. Cell. Physiol. 146: 34–42.
Good DW, Knepper MA and Burg MB (1984) Ammonia and bicarbonate transport by thick ascending limb of rat kidney. Am. J. Physiol. 247: F35-F44.
Glacken MW (1988) Catabolic control of mammalian cell culture. Bio/Technology 6: 1041–1050.
Glacken MW, Adema E and Sinskey AJ et al. (1988) Mathematical description of hybridoma culture kinetics: I. Initial metabolic rates. Biotechnol. Bioeng. 32: 491–506.
Kikeri D, Sun A, Zeidel ML and Herbert SC (1989) Cell membranes impermeable to NH3. Nature 339: 478–480.
Knepper MA, Packer R and Good DW (1989) Ammonium transport in the kidney, Physiol. Reviews 69: 179–249.
Lund P, Brosnan JT and Eggleston LV (1970) The regulation of ammonia metabolism in mammalian tissues. In: W Bartley et al. (eds.), Essays in metabolism, Wiley, New York, pp. 167–180.
Martinelle K and Häggström L (1993) Mechanisms of ammonia and ammonium ion toxicity in animal cells: Transport across cell membranes. J. of Biotechnol. 30: 339–350.
McQueen A and Bailey JE (1991) Growth inhibition of hybridoma cells by ammonium ion: correlation with effects on intracellular pH. Bioproc. Eng. 6: 49–61.
Mercille S and Massie B (1994) Induction of apoptosis in nutrientdeprived cultures of hybridoma and myeloma cells. Biotechnol. Bioeng. 44: 1140–1154.
Newland M, Kamal MN, Greenfield PF and Nielsen LK (1994) Ammonia inhibition of hybridomas propagated in batch, fed-batch, and continuous culture. Biotechnol. Bioeng. 43: 434–438.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Martinelle, K., Westlund, A. & Häggström, L. Ammonium ion transport—a cause of cell death. Cytotechnology 22, 251–254 (1996). https://doi.org/10.1007/BF00353945
Issue Date:
DOI: https://doi.org/10.1007/BF00353945